RNAseq for cultured ctrl and TgAPPsweOCN-Cre osteoblasts from skull bone

Purpose: To investigate how APPswe+ osteoblasts affect skull bone and skull bone marrow remodeling, we isolated tdTomato⁺ calvarial osteoblasts from the skulls of TgAPPswemOCN-Cre;Ai9 mice and control (mOCN-Cre;Ai9) mice at 6-MO using fluorescence-activated cell sorting (FACS) and subjected them to bulk RNA sequencing. Methods: (1) Calvariae from TgAPPswemOCN-Cre;Ai9 mice and control (mOCN-Cre;Ai9) mice were transferred to a dish containing PBS, and the soft tissues were carefully removed using tweezers. The cleaned bones were then cut into small fragments of approximately 1–2 mm² and sequentially digested with collagenase solution (37 °C, 30 min, twice) and trypsin solution (37 °C, 30 min) to remove residual soft tissue and adherent cells. After an additional collagenase digestion, the bone fragments were washed three times with culture medium and transferred to 60 mm culture dishes containing DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 10 mmol/L β-glycerophosphate, and 50 μmol/L L-ascorbic acid-2-phosphate. The culture medium was replaced three times per week. Osteoblasts began to migrate from the bone chips after 3–5 days. After two weeks, the monolayer cells were detached by incubation with trypsin solution and replated onto 100 mm tissue culture dishes in α-MEM supplemented with 10% FBS and 1% penicillin/streptomycin. (2) Total RNA was extracted from purified Td⁺ osteoblasts isolated by flow cytometry from mOCN-Cre; Ai9 and TgAPPswemOCN-Cre; Ai9 mice. RNA integrity was assessed for each sample, and only samples with a RNA Integrity Number (RIN) > 2 were included for further analysis. RNA-seq was performed by BGI America (Cambridge, MA) using the DNBseq platform. Gene expression levels were normalized as fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed genes (DEGs) were identified using DESeq2 and PoissonDis algorithms, with P-values adjusted by the Benjamini–Hochberg (BH) method. Genes with adjusted P ≤ 0.05 and log₂ fold change ≥ 1 were considered significantly differentially expressed. Results: Transcriptomic profiling revealed 425 upregulated and 478 downregulated genes in APPswe-expressing osteoblasts compared to controls. Gene ontology (GO) pathway analysis showed that the upregulated genes were primarily enriched in pathways related to positive regulation of cell migration, cytokine production, Wnt/β-catenin signaling, angiogenesis, cell-cell adhesion, and negative regulation of cell proliferation, while downregulated genes were associated with cell-cell signaling, cell proliferation, and osteoclast differentiation. Conclusions: Early thinning of the skull in TgAPPswemOCN-Cre mice may result from reduced osteoblast proliferation.